Methods to predict cholesterol elevations during immunosuppressant therapy

ABSTRACT

This invention provides methods to predict the degree of elevation of serum cholesterol levels in patients treated with immunosuppressive medication. This invention also provides treatment strategies based on these predictions and kits to carry out these methods.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention belongs to the fields of pharmacology and medicineand provides methods to determine which patients will develop elevatedserum cholesterol levels during treatment with an immunosuppressantdrug. In particular, this invention relates to the use of genomicanalysis to identify patients at risk for developing increasedcholesterol levels during immunosuppressant drug therapy and to methodsto determine optimal treatment strategies for these patients.

2. Description of the Related Art

Immunosuppressant drugs have many important applications in modernmedicine. These drugs are used to suppress the rejection of transplantedorgans, including hearts, lungs and kidneys to prolong the useful lifeof the transplanted organ. In addition, immunosuppressant drugs are usedto treat a wide variety of other diseases such as autoimmune diseases,myocarditis and rheumatoid arthritis. However, the immunosuppressantdrugs have numerous, and sometimes severe, side effects which includecausing cancer and lymphomas and producing a variety of toxic effects oninternal organs, such as the kidney.

Because of the toxic effect of the immunosuppressant drugs, there hasbeen a great deal of effort to develop less toxic alternatives and tofind drugs whose mechanism of action differs from that of the otherimmunosuppressant drugs so that synergistic combinations can be usedwith fewer overall side effects.

Recently an anti-fungal, anti-tumor and immunosuppressive antibioticcalled rapamycin (also known as sirolimus and RAPAMUNE™) has been foundto be effective at inhibiting allograft rejection. Rapamycin has amechanism of action that is unique and markedly different from that ofother immunosuppressant drugs. Rapamycin and its derivatives, such aseverolimus (CERTICAN™)(RAD) act by inhibiting the biochemical pathwaysinvolved in the G1-S phase progression of activated T cells in a Ca²⁺independent manner. See Schuler et al., Transplantation, Vol. 64, pp.36-42 (1997). In this way, rapamycin derivatives such as everolimusblock cytokine signal transduction rather than blocking the productionof cytokines as in the case of other immunosuppressant drugs, such ascyclosporine.

Rapamycin and its derivatives and mycophenolic acid are effectiveimmunosuppressant drugs, however, in some patients the administration ofthese drugs has been found to cause elevations in serum cholesterol andtriglycerides, i.e., hypercholesterolemia and hyperlipidemia. Both ofthese conditions are risk factors for coronary artery disease (CAD) andatherosclerosis in general, especially in diabetic patients.

Hypercholesterolemia itself, is a common condition and can be treatedwith several major classes of drugs. These include the HMG-CoA reductaseinhibitors or the so-called statins, the bile acid-binding resins andnicotinic acid.

Increased serum cholesterol levels during treatment with animmunosuppressant drug, such as rapamycin or its derivatives including,but not limited to, everolimus (CERTICAN™)(RAD) or with mycophenolicacid, is a serious adverse side effect. This is especially true fororgan transplant patients since these patients require long-term(generally life long) treatment. The increase in serum cholesterollevels varies widely from patient to patient and prior to the presentinvention it was not possible to predict which patients would developthese increases. Thus, there is a need for methods to predict whichpatients will experience elevations in serum cholesterol whenimmunosuppressant drugs, such as rapamycin and its derivatives ormycophenolic acid are administered to patients, especially for long-termuse.

SUMMARY OF THE INVENTION

The present invention overcomes this problem by providing a method todetermine the degree of serum cholesterol elevation which will occur ina patient during treatment with an immunosuppressant medicationcomprising: determining for the two copies of the IL-1β gene present inthe patient the identity of the nucleotide pair at the polymorphic site−511 C→T (position 1423 of sequence X04500) of the IL-1β gene; andassigning the patient to a high cholesterol elevation group if bothpairs are AT, assigning the patient to an intermediate cholesterolelevation group if one pair is AT and one pair is GC and assigning thepatient to a low cholesterol elevation group if both pairs are GC.

In a further embodiment this invention provides another method to treata patient with an immunosuppressive medication comprising: determiningfor the two copies of the IL-1β gene present in the patient the identityof the nucleotide pair at the polymorphic site −511 C→T (position 1423of sequence X04500) of the IL-1β gene; and treating the patient with theimmunosuppression medication if both pairs are GC and using alternativetreatment if one pair is AT and one pair is GC or if both pairs are AT.The immunosuppressive medication may be selected from the list in Table2 and may be everolimus. In addition this invention provides that thealternative treatment comprises the addition of a cholesterol-loweringmedication chosen from those listed in Table 1.

In a further embodiment this invention provides a method to determinethe degree of serum cholesterol elevation which will occur in a patientduring treatment with an immunosuppressant medication comprising:determining for the two copies of the IL-1β gene present in the patientthe identity of the nucleotide pair at the polymorphic site −31 T→C(position 1903 of sequence X04500) of the IL-1β gene; and assigning thepatient to a high cholesterol elevation group if both pairs are CG,assigning the patient to an intermediate cholesterol elevation group ifone pair is AT and one pair is GC and assigning the patient to a lowcholesterol elevation group if both pairs are AT.

In a still further embodiment this invention provides a method to treata patient with an immunosuppressive medication comprising: determiningfor the two copies of the IL-1β gene present in the patient the identityof the nucleotide pair at the polymorphic site −31 T→C (position 1903 ofsequence X04500) of the IL-1β gene; and treating the patient with theimmunosuppression medication if both pairs are AT and using alternativetreatment if one pair is AT and one pair is GC or if both pairs are CG.The immunosuppressive medication may be selected from the list in Table2 and may be everolimus. In addition the alternative treatment maycomprise the addition of a cholesterol-lowering medication chosen fromthose listed in Table 1.

In a still further embodiment this invention provides a kit fordetermining the nucleotide pair at the polymorphic site −511 in theIL-1β gene in a patient, comprising: a container containing at least onereagent specific for detecting the nature of the nucleotide pair at thepolymorphic site −511 of the IL-1β gene; and instructions forrecommended treatment options based on the nature of the said nucleotidepair.

In a still further embodiment this invention provides a kit fordetermining the nucleotide pair at the polymorphic site −31 in the IL-1βgene in a patient, comprising: a container containing at least onereagent specific for detecting the nature of the nucleotide pair at thepolymorphic site −31 of the IL-1β gene; and instructions for recommendedtreatment options based on the nature of the said nucleotide pair.

In a further embodiment this invention provides a method to determinethe degree of serum cholesterol elevation which will occur in a patientduring treatment with an immunosuppressant medication comprisingdetermining, for the two copies, containing the IL-1β gene, present inthe patient, the haplotype with regard to the IL-1β gene. The term“haplotype with regard to the IL-1 gene” shall refer to the haplotypeconsisting of the combination of the polymorphisms at the −511 and the−31 position of the IL-1β gene. The patient would be assigned to a highcholesterol elevation group if both chromosomes contain the “highcholesterol” haplotype i.e., T for C at site −511 and C for T at site−31 of the IL-1β gene, and the patient would be assigned to anintermediate cholesterol elevation group if one chromosome contains the“high cholesterol” haplotype and one contains the “low cholesterol”haplotype and the patient would be assigned to a low cholesterolelevation group if both chromosomes contain the “low cholesterol”haplotype, i.e. C at site −511 and T at site −31.

In a still further embodiment this invention provides a method to treata patient with an immunosuppressive medication comprising determining,for the two chromosomes containing the IL-1β gene, present in thepatient, the haplotype with regard to the IL-1β gene, and treating thepatient with the immunosuppression medication if both chromosomescontain the “low cholesterol” haplotype or if one chromosome containsthe “low cholesterol” haplotype and one contains the “high cholesterol”haplotype and using alternative treatment if both chromosomes containthe “high cholesterol” haplotype. The immunosuppressive medication maybe selected from the list in Table 2 and may be everolimus. Thealternative treatment may comprise the addition of acholesterol-lowering medication chosen from those listed in Table 1.

In a further embodiment this invention provides methods of determiningthe identity of the nucleotide pair at the site −511 and −31 of theIL-1β gene in a patient or the haplotype of the IL-1β gene in a patientby finding SNPs anywhere in the chromosome which are in linkagedisequilibrium with the −511 polymorphism or the −31 polymorphism in theIL-1β gene and using the relationship of the said SNP or SNPs todetermine the nature of the nucleotide pair or haplotype of interest andusing this information to estimate cholesterol elevation during IMtherapy and to make treatment decisions.

A further embodiment of this invention is a kit for determining thenature of the haplotype of the IL-1β gene which includes; a containercontaining at least one reagent specific for detecting the nature of thenucleotide pair at the polymorphic site −511 of the IL-1β gene; and acontainer containing at least one reagent specific for detecting thenature of the nucleotide pair at the polymorphic site −31 of the IL-1βgene; and instructions for determining the haplotype from the results ofthe above and instructions for recommended treatment options based onthe nature of the indicated haplotype.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: LS mean total cholesterol levels compared to the (−511) IL-1βCC, CT or TT genotypes in all treatment groups combined within the RADB251 clinical trial.

FIG. 2: LS mean total cholesterol levels compared to the (−31) IL-1β CC,CT or TT genotypes in all treatment groups combined within the RAD B251clinical trial.

FIG. 3: LS mean HDL cholesterol levels compared to the (−511) IL-1β CC,CT or TT genotypes in all treatment groups combined within the RAD B251clinical trial.

FIG. 4: LS mean HDL cholesterol levels compared to the (−31) IL-1β CC,CT or TT genotypes in all treatment groups combined within the RAD B251clinical trial.

FIG. 5: LS mean LDL cholesterol levels compared to the (−511) IL-1β CC,CT or TT genotypes in all treatment groups within the RAD B251 clinicaltrial.

FIG. 6: LS mean LDL cholesterol levels compared to the (−31) IL-1β CC,CT or TT genotypes in all treatments groups within the RAD B251 clinicaltrial.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods to determine the degree of serumcholesterol elevation that will occur in a patient during treatment withan immunosuppressant medication (IM), such as rapamycin or itsderivatives.

In one embodiment, a patient who is a potential candidate for treatmentwith an IM would have blood drawn for a determination of the presence ofa polymorphism, i.e., cytosine (C)→thymine (T) at nucleotide position−511 (in the promoter region with no amino acid change); this is a C→Tchange at nucleotide position 1423 of sequence X04500, in the two copiesof the interleukin-1-beta (IL-1β) gene present in the patient. If thenucleotide pair at position −511 is AT in both copies of the gene, thenthe patient will experience a high elevation in serum cholesterol levelsduring treatment with an IM.

If the nucleotide pair at position −511 is AT in one copy and CG in theother copy, then the patient will have an intermediate elevation intheir cholesterol levels during treatment with an immunosuppressantmedication.

If the nucleotide pair is GC at both copies at position −511, then thepatient will have a low elevation in serum cholesterol levels duringtreatment with an IM.

In another embodiment, a determination of which medications to use totreat a patient in need of treatment with an IM would be based on theresults of the determination of the nature of the nucleotide pairs atposition −511 in the IL-1β gene present in the patient.

If both nucleotide pairs are GC then the patient would be treated withan IM. This immunosuppressant medication could be any of those shown inTable 2, including rapamycin or one of its derivatives, including, butnot limited to, everolimus (Certican™)(RAD).

If both nucleotide pairs are AT, or if one pair is AT and one pair isGC, then the patient would be treated with an alternative medicationthat did not raise cholesterol levels or alternatively the patient wouldbe treated with a cholesterol-lowering drug in addition to the IM andthe patients' cholesterol levels would be monitored during treatment.This cholesterol-lowering drug, which would be used in combination withIM, could be one or more of the medications chosen from the list inTable 1 below.

In a further embodiment, a patient who is a potential candidate fortreatment with an IM would have blood drawn for a determination of thepresence of a polymorphism T→C at nucleotide position −31 (in thepromoter region with no amino acid change); this is a T→C change atnucleotide position 1903 of sequence X04500 in the two copies of theIL-1β gene present in the patient. If the nucleotide pair at position−31 is a CG in both copies of the gene, then the patient will experiencea high elevation in serum cholesterol levels during treatment with anIM. If the nucleotide pair at position −31 is AT in one copy and CG inthe other copy, then the patient will have an intermediate elevation intheir cholesterol levels during treatment with an IM. If the nucleotidepair is AT at both copies at position −31, then the patient will have alow elevation in serum cholesterol levels during treatment with an IM.

In another embodiment, a determination of which medications to use totreat a patient in need of treatment with an IM would be based on theresults of the determination of the nature of the nucleotide pairs atposition −31 in the IL-1 gene present in the patient. If both nucleotidepairs are AT then the patient would be treated with an IM.

This IM could any of those shown in Table 2 including, but not limitedto, rapamycin or one of its derivatives including, but not limited to,everolimus (CERTICAN™) (RAD).

If both nucleotide pairs are GC or if the nucleotide pair is AT in onecopy and CG in the other copy, then the patient would be treated with analternative medication that did not raise cholesterol levels oralternatively the patient would be treated with a cholesterol-loweringdrug in addition to the IM and the patients' cholesterol levels would bemonitored during treatment. This cholesterol-lowering drug, which wouldbe used in combination with IM, could be one or more of the medicationschosen from the list in Table 1 below.

Suitable rapamycins for use in the methods of this invention are, e.g.,as described in U.S. Pat. Nos. 3,929,992 and 5,258,389 and in WO94/09010 and WO 01/60345, all of which are hereby incorporated byreference in their entirety and for all purposes. TABLE 1 AntilipemicAgents (Cholesterol-Lowering Drugs) Bile Acid Sequestrants Colestipol(COLESTID ™ Pharmacia & Upjohn) Fibric Acid Derivatives Clofibrate(ATROMID-S ™ Wyeth-Ayerst) Gemfibrozil (LOPID ™ Park-Davis) Fenofibrate(TRICOR ™ Abbott) HMG-CoA Reductase Inhibitors Fluvastatin (LESCOL ™Novartis) Atorvastatin (LIPITOR ™ Parke-Davis) Lovastatin (MEVACOR ™Merck) Pravastatin (PRAVACOL ™ Bristol-Myers Squibb) Simvastatin(ZOCOR ™ Merck) Nicotinic Acid Niacin (NIASPAN ™ Kos)

TABLE 2 Immunosuppressant Drugs Rapamycin (sirolimus, RAPAMUNE ™)Everolimus (CERTICAN ™) (RAD) Mycophenolic acid and MycophenolateMofetil (CELLCEPT ™) (MMF) Azathioprine (IMURAN ™) Cyclosporine(NEORAL ™) Tacrolimus (PROGRAF ™)

The following example is provided for the purpose of furtherillustration only and is not intended to be a limitation on thedisclosed invention.

EXAMPLE 1

The RAD B251 Study

Overall Study Design

The RAD B251 study was a randomized, multicenter, double-blind, parallelgroup study of the efficacy and safety of everolimus (Certican™) (RAD)versus mycophenolate mofetil (MMF) used in combination with cyclosporine(CsA) (NEORAL®) and prednisone. The study consisted of three periods: aScreening period, a Baseline period and a Double-Blind Treatment period.Following the Baseline assessments, patients who meet theinclusion/exclusion criteria were randomized into one of the threetreatment groups (1:1:1) once it has been ascertained that the allograftis functional and that oral medication can be tolerated (within 48 hourspost-transplantation). Determination of allograft function was by theinvestigator's judgement and was based upon adequate urine output andevidence of falling creatinine levels. The day of randomization andadministration of the first dose of study medication was recorded as Day1 of the study. The three treatment groups are described below:

Dose Level 1: 0.75 mg RAD bid+NEORAL®+prednisone

Dose Level 2: 1.5 mg RAD bid+NEORAL®+prednisone

Comparator 1 g MMF bid+NEORAL®+prednisone

Concomitant Therapy

Initiation and Maintenance of NEORAL®

Oral CsA (NEORAL®) administration was begun at 6-12 mg/kg/day p.o. andwas adjusted to maintain a 12-hour trough level reflecting a standardtarget assay range. Intravenous (i.v.) administration of CsA was avoidedunless mandated by the clinical situation. Whole blood levels werebrought into the therapeutic ranges listed below as rapidly as possible.Once this was achieved, doses of CsA were only adjusted for maintainingtrough blood levels within the target ranges.

Weeks 1-4: 200-350 ng/mL

Months 2-36: 100-300 ng/mL

On the days when blood for CsA or RAD measurements was to be drawn, thepatient received the prior dose of NEORAL® and study medication 12±1hour prior to the blood draw. The patients were instructed to adjusttheir medication schedule on the day previous to the blood draw toachieve proper timing and the exact time of administration of theevening dose was recorded. Study medication and NEORAL® due on the dayof the blood draw were not to be taken by the patient, but were broughtto the clinic and taken after the blood draw was completed.

Prednisone

Immediately prior to transplant, patients could receive up to 1 gmethylprednisolone i.v. and then up to 500 mg methylprednisolone i.v. 12hours later. As soon as possible post-transplantation, oral prednisonewas initiated at 0.35-2.0 mg/kg/day and tapered in order to achieve adose of 20 mg/day, or 0.25 mg/kg/day, by Day 30 and of no less than 5mg/day for the first six months.

Cytomegalovirus (CMV) Prophylaxis

CMV prophylaxis was mandatory for all cases in which the donor testspositive and the recipient tests negative for CMV. Treatment withganciclovir, CMV hyperimmune globulin or acyclovir was permitted and wasadministered according to local practice. All cases other than CMVpositive donors to CMV negative recipients were treated according tolocal practice. CMV prophylaxis was also recommended following anyantibody treatment of acute rejection episodes.

PCP Prophylaxis

All patients were also started on trimethoprim-sulfamethoxazole, onesingle strength tablet per day, starting when oral medication could betolerated and continuing for the first six months after transplantation.Dosage was decreased at the investigator's discretion to one singlestrength tablet 3 times a week for the second six months. Treatmentafter one year was according to local practice. Aerosolized pentamidineor dapsone was administered to patients unable to toleratetrimethoprim-sulfamethoxazole.

Other Concomitant Therapy

No medication other than the study drugs, study prophylaxis and theusual medications of the patients were given during the full treatmentperiod of the study, i.e., from the initial day of screening until allof the final study evaluations have been completed. Exceptions to thisrule applied only to medications that were needed to treat adverseevents (AEs). The administration of any additional medication (includingover-the-counter medications and vitamins) were clearly documented onthe Prior and Concomitant Medications Case Report Form (CRF). Ifrequired for an AE, concomitant medications were clearly documented andcross-referenced on the AEs CRF.

All immunosuppressive drugs other than those specified by protocol weredisallowed. Permissible anti-rejection therapy includesmethylprednisolone and anti-lymphocyte antibody therapy according to theguidelines in “Treatment of Acute Rejection Episodes”. Patientsrequiring tacrolimus or MMF for rescue therapy were discontinued fromstudy drug. Terfenadine, astemizole and cisapride were prohibited whilethe patient is on study medication. The use of phenobarbital, phenytoin,carbamazepine, or ketoconazole was strongly discouraged.

A treatment period of three years followed transplantation. During theDouble-Blind Treatment period, the patients were seen at Days 7, 14 and28 and at Months 2, 3, 6, 9, 12, 18, 24, 30 and 36. Renal biopsies wererequired at Baseline (may be intra-operative) and at the time of anysuspected rejection. Blinded study drug administration ceased at 3years.

Male and female patients, 16-65 years of age who are scheduled toundergo primary cadaveric, living unrelated or non-HLA identical relateddonor kidney transplantation, were allowed to enter the study. Patientswho discontinued the study prematurely were not replaced. Each patienthad to meet all of the inclusion/exclusion criteria to be eligible forentry into the study.

The RAD B251 study was designed to assess the safety and efficacy of twooral doses of RAD compared to MMF in de novo renal transplant recipientsas measured by the incidence of biopsy proven acute allograft rejectionepisodes, graft loss or death. MMF was chosen as the comparator agentbecause of its widespread use in renal transplantation.Pharmacogenetics Analysis

In an effort to identify genetic factors that are associated withincreased cholesterol and lipid levels observed in patients treated witheverolimus (RAD), 47 single nucleotide polymorphisms (SNPs) from 24genes within genomic polydeoxribonucleotide (DNA) of patientsparticipating in the RAD B251 clinical trials were examined. Of the 47SNPs that were examined, 21 were experimentally determined to be notpolymorphic. Of the 26 that were polymorphic, two SNPs in the IL-1β genepromoter at positions (−511) and (−31) showed statistical significancein relation to changes in cholesterol levels in patients participatingin the RAD B251 clinical trial.

Patients who were homozygous for the IL-1β (−511) C→T base transition(T-T) or the IL-1β (−31) T→C base transition (C-C) had the highest leastmean levels of total cholesterol at their last visit regardless oftreatment received during the study (p=0.0018 and p=0.0013respectively). (The values on the figures, etc. refer to the absoluteserum cholesterol levels at the last visit, however during thestatistical analysis this value was defined as the dependent variableand the cholesterol level at baseline as the independent variable thusautomatically taking into consideration the baseline level).

The increase in total cholesterol levels was due to both increasedlevels of HDL and LDL: patients homozygous for the T allele at the(−511) position or the C allele at the (−31) position had the highestleast square mean levels of HDL (p=0.0214 and p=0.0514 respectively) andLDL (p=0.0159 and p=0.0091 respectively) at their last visit.Importantly, however, the HDL to LDL ratios remained the same regardlessof genotype.

Therefore, our findings suggest that individuals homozygous for the Tallele at position (−511) and homozygous for the C allele at position(−31) of the IL-1β gene promoter may be predisposed to larger increasesin total blood cholesterol levels upon treatment with either theRAD/NEORAL® or MMF/NEORAL® regimens.

Methods

Samples

A total of 82 unique samples from the RAD B251 clinical trial weregenotyped upon their consent to participate in the pharmacogeneticevaluation. This represents about 15% of the total population thatparticipated in the RAD B251 clinical trial. Blood samples from eachpatient were collected at the individual trial sites and then shipped toCovance (Geneva, Switzerland). The genomic DNA of each patient wasextracted from the blood by Covance using the PUREGENE™ DNA IsolationKit (D-50K) (Gentra, Minneapolis, Minn.).

Genotyping

A total of 47 unique polymorphisms corresponding to 24 genes wereanalyzed for each clinical trial. Candidate genes involved in metabolismof the drug, hypercholesterolemia, hyperlipidemia, immunosuppression andinflammation were chosen for this study. SNP assays were designed usinginformation from public databases, such as OMIM, the SNP Consortium,Locus Link and dbSNP, and the Third Wave Technologies, Inc. (TWT,Madison, Wis.) website (http://64.73.25.65:8080/coe/index.jsp). Theresulting probe sets for the genotyping assay were generated by TWT.Genotyping was performed with 60 ng of genomic DNA using the INVADER®assay developed by TWT (9-10) according to the manufacturer'sinstructions. See Lyamichev et al., Nat Biotechnol., Vol. 17, pp.292-296 (1999); and Ryan, Mol. Diagn., Vol. 4, pp. 135-144 (1999).

Polymerase Chain Reaction (PCR) for the (−511) IL-1β SNP was performedin a 20 μL reaction containing: 10-70 ng genomic DNA, 160 μM dNTPs, 10mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl₂, 0.6 μMIL-1β(−511)-forward primer, 0.6 μM IL-1β(−511)-reverse primer and 0.03 UTaq DNA polymerase (Applied Biosystems, Foster City, Calif.). Thirty-six(36) rounds of amplification were performed using the followingconditions: 94° C., 30 seconds; 55° C., 30 seconds; 72° C., 30 seconds.Nine samples were then fractionated (5 μL) on a 2% agarose gel andvisualized by ethidium bromide staining to confirm amplification. A 1:7dilution of the PCR product was run against TWT SNP#128069 using a384-well biplex plate for amplified DNA.

Primer sequences are as follows: (SEQ ID No.1) IL-1β(-511)-forward5′-GCAGAGCTCATCTGGCATTG-3′; (SEQ ID No.2) IL-1β(-511)-reverse5′-TATGTGGGACAAAG TGGAAG-3′.

PCR for the (−31) IL-1β SNP was performed in a 25 μL reactioncontaining: 1 ng genomic DNA, 40 μM dNTPs, 10 mM Tris-HCl [pH 8.3], 50mM KCl, 1.5 mM MgCl₂, 0.75 μM IL-1β(−31)-forward primer, 0.75 μMIL-1β(−31)-reverse primer and 0.15 U Gold Taq DNA polymerase (AppliedBiosystems, Foster City, Calif.). Thirty-eight (38) rounds ofamplification were performed using the following conditions: 94° C., 30seconds; 58° C., 30 seconds; 72° C., 30 seconds. All samples were thenfractionated (5 μL) on a 2% agarose gel and visualized by ethidiumbromide staining to confirm amplification.

Primer sequences are as follows: (SEQ ID No.3) IL-1β(-31)-forward5′-GCACAACGATTGTCAGGAAAAC-3′; (SEQ ID No.4) IL-1β(-31)-reverse(5′-ATGCATACA CACAAAGAGGCAG-3′.

A 1:10 dilution of the PCR product was run against TWT SNP# 274339 forRAD B251 using a 384-well biplex plate for amplified DNA. RFLP analysiswas used to determine genotypes using the Alu-I restriction enzyme (NewEngland Biolabs, Beverly, Mass.). RFLP digests were performed in a 20 μLreaction containing: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT[pH 7.9], 8 ng amplified genomic DNA and 0.5 mM Alu-I enzyme. Allsamples were incubated for 17 hours at 37° C. and then fractionated (19μL) on a 3% agarose gel and visualized by ethidium bromide staining todetermine band size. Nucleotide sequence surrounding the (−511)IL-1β polymorphism Surrounding Gene Position Allele 1 Allele 2 SequenceIL-1β −511 C T CTGCAATTGACAGAGAGCT CC[C,T]GAGGCAGAGAACAGCACCCAAGGTAGAGACC CA Allele 1 (SEQ ID No. 5);CTGCAATTGACAGAGAGCTCC[C]GAGGCAGAGAACAGCACCCAAGGTAG AGACCCA Allele 2 (SEQID No. 6); CTGCAATTGACAGAGAGCTCC[T]GAGGCAGAGAACAGCACCCAAGGTAG AGACCCANucleotide sequence surrounding the (−31) IL-1β polymorphism SurroundingGene Position Allele 1 Allele 2 Sequence IL-1β −31 C TTCCTACTTCTGCTTTTGAA AGC[T,C]ATAAAAACAGC GAGGGAGAAACTGGCAGAT ACCAAACCTCAllele 1 (SEQ ID No. 7)TCCTACTTCTGCTTTTGAAAGC[C]ATAAAAACAGCGAGGGAGAAACTGG CAGATACCAAACCTCAllele 2 (SEQ ID No. 8)TCCTACTTCTGCTTTTGAAAGC[T]ATAAAAACAGCGAGGGAGAAACTGG CAGATACCAAACCTCStatistical Analysis

An analysis of covariance model was used for the analysis of the effectof genotype and treatment on cholesterol levels using the 24-monthlab_b.sd2 RADB 251 clinical data set. Terms in the model include thefinal cholesterol level, the initial cholesterol level as the covariant,and the genotype and treatment as the main effectors. The odds ratios,95% confidence limits, and Chi-square analysis were calculated whereapplicable. All statistical analyses were performed using the SAS 8.02software. To correct for multiple testing, the Bonferroni correctionmethod was performed.

Results

In the RAD B251 study, 47 unique SNPs corresponding to 24 genes weregenotyped for each patient consenting to pharmacogenetics analysisparticipating in the RAD B251 clinical trial. A comparison of thepatients that consented to pharmacogenetics analysis to the overallpatient distribution for each respective clinical trial is shown inTable 3. TABLE 3 RAD B251: Distribution of Pharmacogenetic SamplesCompared to the Overall Clinical Trial Samples Pharmacogenetic Trialsamples Samples Age (years) 43.34 43.49 Race Caucasian (61) 74% (388)68%   Black  (9) 11% (93) 16%  Oriental (0) 0% (11) 2%   Other (12) 15%(76) 13%  Gender Male (51) 62% (357) 63%   Female (31) 38% (211) 37%  Treatment RAD001(1.5 mg/d)   (24) 29.3% (189) 33.3% RAD001(3.0 mg/d)  (29) 35.3% (189) 33.3% MMF (2 g/d) (29) 35% (190) 33.4% Weight (kg)80.7 77.1 Baseline CHO (mg/dL) 168.7 162.8 HDL (mg/dL) 40.9 40.8 LDL(mg/dL) 96.6 96.8 TGC (mg/dL) 155.3 119.9 End of treatment CHO (mg/dL)234.5 232.2 HDL (mg/dL) 52.7 53.3 LDL (mg/dL) 123.2 126.6 TGC (mg/dL)283.0 254.6

Only one statistically significant difference was found between thepatient population used in the pharmacogenetic study compared to theoverall patient population in the RAD B251 clinical trial: thedifferences in the mean TGC values among the pharmacogenetic patentpopulation and the overall RAD B251 patient population were found to bestatistically significant (p<0.001). This comparison therefore suggeststhat the patient population used in the pharmacogenetic study isrepresentative of the overall patient population tested in each trial.Of the 47 SNPs that were tested in this study, 21 were experimentallydetermined to be not polymorphic. Therefore, 26 SNPs were used in theanalysis described below.

Statistical analysis of the genotypes with the RAD B251 clinical dataset identified a polymorphism within the IL-1β gene promoter at position(−511) that had a significant association with cholesterol levels. Asshown in Table 4 and FIG. 1, the IL-1β (−511) (TT) genotype in patientsfrom both treatment groups together correlated with the highest increasein levels of total cholesterol measured at their last visit (p=0.0018).TABLE 4 LS Mean Total Cholesterol Levels (mg/dL) by (−511) IL-1β CC, CTor TT Genotypes and Treatment Groups Within the RAD B251 Clinical TrialRAD and MMF RAD MMF treatment groups (1.5 and 3.0 mg/day) (2 mg/day)combined Genotype CC CT TT CC CT TT CC CT TT No. of patients 20 25 9 129 7 32 34 16 LS means 217.3 242.9 290.3 200.9 229.6 254.1 211.4 239.5272.9 P-value 0.0125 0.0125 0.0125 0.0866 0.0866 0.0866 0.0018 0.00180.0018

A similar association was observed for the IL-1 (−31) (C-C) genotype(p=0.0013) (Table 5 and FIG. 2). TABLE 5 LS Mean Total CholesterolLevels (mg/dL) by (−31) IL-1β CC, CT or TT Genotypes and TreatmentGroups Within the RAD B251 Clinical Trial RAD and MMF RAD MMF treatmentgroups (1.5 and 3.0 mg/day) (2 mg/day) combined Genotype CC CT TT CC CTTT CC CT TT No. of patients 9 26 19 7 9 12 16 35 31 LS means 291.1 240.4216.6 254.1 239.6 200.9 272.9 239.5 211.4 P-value 0.009 0.009 0.0090.0625 0.0625 0.0625 0.0013 0.0013 0.0013

To analyze this correlation further, it was tested whether anassociation existed between the IL-1β (−511) genotype and levels of HDLand LDL. As shown in FIG. 3 and Table 6, the IL-1β (−511) (T-T) genotypein patients from both treatment groups together correlated with thehighest levels of HDL measured at their last visit (p=0.0214). TABLE 6LS Mean HDL levels (mg/dL) by (−511) IL-1β CC, CT or TT Genotypes andTreatment Groups Within the RAD B251 Clinical Trial RAD and MMF RAD MMFtreatment groups (1.5 and 3.0 mg/day) (2 mg/day) combined Genotype CC CTTT CC CT TT CC CT TT No. of patients 20 25 9 12 9 7 32 34 16 LS means47.6 54.9 68.4 45.5 56.8 50.4 47.8 54.4 58.9 P-value 0.0164 0.01640.0164 0.0819 0.0819 0.0819 0.0214 0.0214 0.0214

It has been previously reported that the IL-1β (−511) polymorphism is instrong linkage disequilibrium to another polymorphism in the IL-1βpromoter at position (−31). See El-Omar et al., Nature, Vol. 404, pp.398-402 (2000). In this study, 254 alleles were tested in patientsconsenting to pharmacogenetic analysis in the RAD B251 clinical trial.It is reported that the IL-1β (−511) and (−31) polymorphisms are in99.2% linkage disequilibrium. Therefore, a similar association wouldoccur with the IL-1β (−511) polymorphism and cholesterol levels could bedetected as with the IL-1β (−31) polymorphism. Statistical analysis ofthe RAD B251 clinical data set to the IL-1β (−31) polymorphismidentified a significant association with cholesterol levels. As shownin Table 5 and FIG. 2, the IL-1β (−31) (C-C) genotype in patients fromboth treatment groups together correlated with the highest increase inlevels of total cholesterol measured at their last visit (p=0.0013). Toanalyze this correlation further, it was tested whether an associationexisted between the IL-1β (−31) genotype and levels of HDL and LDL. Asshown in FIG. 4 and Table 7, the IL-1β (−31) (C-C) genotype in patientsfrom both treatment groups together weakly correlated with the highestlevels of HDL measured at their last visit (p=0.0514). TABLE 7 LS MeanHDL Levels (mg/dL) by (−31) IL-1β CC, CT or TT Genotypes and TreatmentGroups Within the RAD B251 Clinical Trial RAD and MMF RAD MMF treatmentgroups (1.5 and 3.0 mg/day) (2 mg/day) combined Genotype CC CT TT CC CTTT CC CT TT No. of patients 9 26 19 7 9 12 16 35 31 LS means 65.1 5548.4 50.4 56.8 45.5 58.9 54.4 47.8 P-value 0.0205 0.0205 0.0205 0.18930.1893 0.1893 0.0514 0.0514 0.0514

A similar correlation was identified with LDL levels as well (p=0.0159,FIG. 5 and Table 8). TABLE 8 LS Mean LDL Levels (mg/dL) by (−511) IL-1βCC, CT or TT Genotypes and Treatment Groups Within the RAD B251 ClinicalTrial RAD and MMF RAD MMF treatment groups (1.5 and 3.0 mg/day) (2mg/day) combined Genotype CC CT TT CC CT TT CC CT TT No. of patients 2025 9 12 9 7 32 34 16 LS means 112.5 123.6 144.5 113.9 118.6 143.7 110.5123.8 145.8 P-value 0.1646 0.1646 0.1646 0.2848 0.2848 0.2848 0.01590.0159 0.0159

A stronger correlation was identified with LDL levels and the −31 IL-1βpolymorphism (p=0.0091, FIG. 6 and Table 9). TABLE 9 LS Mean LDL Levels(mg/dL) by (−31) IL-1β CC, CT or TT Genotypes and Treatment GroupsWithin the RAD B251 Clinical Trial RAD and MMF RAD MMF treatment groups(1.5 and 3.0 mg/day) (2 mg/day) combined Genotype CC CT TT CC CT TT CCCT TT No. of patients 9 26 19 7 9 12 16 35 31 LS means 145.1 124.1 112.2143.1 119.8 107.8 145.9 124.5 108.5 P-value 0.143 0.143 0.143 0.20610.2061 0.2061 0.0091 0.0091 0.0091

Importantly, the HDL to LDL ratios remained unchanged between genotypegroups. The findings presented in this study would predict a greaterlikelihood of individuals with a certain allele to experience persistentincreases in cholesterol levels upon treatment with the RAD/NEORAL®regimen than individuals that do not possess the allele. A similar trendwas identified in individuals treated with the MMF/NEORAL® regimen, butthe results did not meet the p=0.05 statistical significance.

Since total blood cholesterol levels ≧240 mg/dL are generally consideredto be excessively elevated, it was therefore decided to determine theodds ratio for a patient with the IL-1β (−511) or IL-1β (−31)polymorphisms to encounter an increase in total blood cholesterol levelsresulting in a final concentration ≧240 mg/dL after being treated withthe RAD/NEORAL® or MMF/NEORAL® regimens. See, Cecil Textbook ofMedicine, Goldman and Bennett, editors, Saunders, 6^(th) Edition (2000).

As shown in Table 10 below, the odds ratio indicates that patients are5.67 (95% confidence limits: 1.20-9.01) times more likely to have anincrease in total blood cholesterol levels to a final concentration ≧240mg/dL when treated with the RAD/NEORAL® regimen if they contain a T atposition (−511) in the IL-1β gene promoter, or 7.23 (95% confidencelimits: 1.20-9.01) times more likely to have an increase in total bloodcholesterol levels to a final concentration ≧240 mg/dL when treated withthe RAD/NEORAL® regimen if they contain a C at position (−31) in theIL-1β gene promoter. These findings are statistically significant(p=0.0207 and p=0.0096, respectively) and could be used as aprecautionary measure in the treatment of transplantation patients withthe RAD/NEORAL® regimen since hypercholesterolemia is easily treatable.TABLE 10 Odds Ratio for the (−511) and (−31) IL-1β Genotypes andCholesterol Levels (−511) IL-1β Polymorphism Obs. Genotype Exp. CT-TT CCTotal >239 mg/dL 24 7 31 18.90 12.10 ≦239 mg/dL 26 25 51 31.10 19.90 5032 82 Odds ratio = 5.67 (95% CI: 1.20-9.01) p = 0.0207 (Fisher's Exacttest) (−31) IL-1β Polymorphism Obs. Genotype Exp. CC-CT TT Total >239mg/dL 25 6 31 19.28 11.72 ≦239 mg/dL 26 25 51 31.72 19.28 51 31 82 Oddsratio = 7.23 (95% CI: 1.20-9.01) p = 0.0096 (Fisher's Exact test)Bonferroni Correction for Multiple Testing

A correction factor is needed due to the number of SNPs that wereanalyzed in this study. To do so, the Bonferroni correction method wasperformed which dictated a p-value of 0.0019.${Bonferroni} = {\frac{0.05}{\eta} = {\frac{0.05}{26} = 0.0019}}$η = RAD_number_of_tests

Therefore, the finding between the IL-1β (−511) and IL-1β (−31)polymorphisms and total cholesterol levels (p=0.0018 and p=0.0013,respectively) is still be considered as significant.

Linkage Disequilibrium of the (−511) and (−31) IL-1β SNPs

It has been reported that the IL-1β (−511) C→T polymorphism is in stronglinkage disequilibrium (99.5%) with another polymorphism within theIL-1β promoter located at position (−31) that results in a T→C basetransition. See El-Omar et al., Nature, Vol. 404, pp. 398-402 (2000).Therefore, it is predicted that patients with a T at position (−511) ofthe IL-1β promoter would have a C at position (−31). This finding isconfirmed in the patients tested in these two trials. In the wild-typeIL-1β gene, T is found at position at −31. This T is very important forthe expression of IL-1β because it is part of the TATA box sequence(TATAAAA) which plays a critical role in the transcriptional initiationof IL-1β. In general, TATA box sequences are involved in recruiting andpositioning the transcriptional machinery at the correct position withingenes to ensure that transcription begins at the correct place. The T→Cpolymorphism at position (−31) would disrupt this important TATA boxsequence (TATAAAA to CATAAAA), thus making it inactive and prohibitingthe efficient initiation of transcription of the IL-1β gene. The lack ofbinding of the transcriptional machinery to this altered IL-1β TATA boxsequence has been shown. See El-Omar, supra.

Therefore, the existence of any other polymorphism which is in linkagedisequilibrium with either the polymorphism within the IL-1β promoter(located at position (−31) that results in a T→C base transition) or thepolymorphism located at −511 (C→T) of the IL-1β promoter, would alsohave a predictive effect on the degree of cholesterol elevation expectedin a patient during treatment with an IM. The means for thedetermination of other polymorphisms which are in linkage disequilibriumwith the (−31) polymorphism is well known to one of skill in the art.Any such polymorphism, now known or discovered in the future, could beused in the methods of this invention to predict the degree of likelycholesterol elevation in patients treated with an IM or to helpdetermine treatment choices for such patients.

Biological Significance of the Findings

The IL-1β (−31) (C-C) genotype has clinical relevance. IL-1β has beenshown to inhibit cholesterol biosynthesis by 25%. See El-Omar et al.,supra. Therefore, the result of this polymorphism would mean thatpatients with the IL-1β (−31) (C-C) genotype, corresponding to the IL-1β(−511) (T-T) genotype, would have decreased levels of IL-1β, therebylosing the inhibition of cholesterol biosynthesis by IL-1β, resulting inelevated levels of cholesterol in the blood. This type of finding wasobserved in the RAD B251 trial. As shown in Tables 4 and 5 and FIGS.1-2, patients with the IL-1β (−511) (T-T) genotype and IL-1β (−31) (C-C)genotype had the highest least square mean levels of total cholesterol,regardless of treatment.

It has also been reported that IL-1β increases LDL receptor geneexpression through the activation of the extracellular signal-regulatedkinases (ERKs). See Kumar et al., J. Biol. Chem., Vol. 273, pp.15742-15748 (1998).

Elevations in LDL receptor expression would result in an increase in theamount of cholesterol that is internalized into cells, thereby loweringtotal cholesterol levels in the blood. This has relevance to RAD(everolimus) since this drug has been shown to inhibit biochemicalpathways that are required for cell progression through late G1 andentry into S. Importantly, the ERKs have been shown to be involved inthis process. Therefore, it is possible that ERK activity would bedecreased by everolimus. Because everolimus would inhibit ERK activity,LDL receptor expression would decrease in all patients, independently ofIL-1β expression, thereby causing increased levels of LDL and thus totalcholesterol in patients taking everolimus. It is unlikely thateverolimus completely inhibits ERK activation. Therefore, patients withthe IL-1β (−511) (T-T) and IL-1β (−31) (C-C) genotypes would be ableinduce some expression of the LDL receptor. However, those patientswould have very low levels of IL-1β, and therefore less LDL receptorexpression, thereby resulting in lower amounts of cholesterol beinginternalized into cells and elevating blood cholesterol levels. Thisexplanation would thus account for the highest levels of cholesterolobserved in patients with the IL-1β (−31) (C-C) genotype. Significantly,patients with the IL-1β (−511) (T-T) genotype, corresponding to theIL-1β (−31) (C-C) genotype, had the significantly higher levels of LDL(p=0.0159) as compared to patients with other IL-1β genotypes (FIG. 3and Table 4).

Identification and Characterization of SNPs

Many different techniques can be used to identify and characterize SNPs,including single-strand conformation polymorphism analysis, heteroduplexanalysis by denaturing high-performance liquid chromatography (DHPLC),direct DNA sequencing and computational methods. See Shi, Clin. Chem.,Vol. 47, pp. 164-172 (2001). Thanks to the wealth of sequenceinformation in public databases, computational tools can be used toidentify SNPs in silico by aligning independently submitted sequencesfor a given gene (either cDNA or genomic sequences). Comparison of SNPsobtained experimentally and by in silico methods showed that 55% ofcandidate SNPs found by SNPFinder(http://lpgws.nci.nih.gov:82/perl/snp/snp_cgi.pl) have also beendiscovered experimentally. See, Cox et al., Hum. Mutal., Vol. 17, pp.141-150 (2001). However, these in silico methods could only find 27% oftrue SNPs.

The most common SNP typing methods currently include hybridization,primer extension and cleavage methods. Each of these methods must beconnected to an appropriate detection system. Detection technologiesinclude fluorescent polarization, (see Chan et al., Genome Res., Vol. 9,pp. 492-499 (1999)), luminometric detection of pyrophosphate release(pyrosequencing), (see Ahmadiian et al., Anal. Biochem., Vol. 280, pp.103-110 (2000)), fluorescence resonance energy transfer (FRET)-basedcleavage assays, DHPLC, and mass spectrometry (see Shi, Clin. Chem.,Vol. 47, pp. 164-172 (2001) and U.S. Pat. No. 6,300,076 B1). Othermethods of detecting and characterizing SNPs are those disclosed in U.S.Pat. Nos. 6,297,018 B1 and 6,300,063 B1. The disclosures of the abovereferences are incorporated herein by reference in their entirety.

In a particularly preferred embodiment the detection of the polymorphismcan be accomplished by means of so called INVADER™ technology (availablefrom Third Wave Technologies Inc. Madison, Wis.). In this assay, aspecific upstream “invader” oligonucleotide and a partially overlappingdownstream probe together form a specific structure when bound tocomplementary DNA template. This structure is recognized and cut at aspecific site by the Cleavase enzyme, and this results in the release ofthe 5′ flap of the probe oligonucleotide. This fragment then serves asthe “invader” oligonucleotide with respect to synthetic secondarytargets and secondary fluorescently-labeled signal probes contained inthe reaction mixture. This results in specific cleavage of the secondarysignal probes by the Cleavase enzyme. Fluorescence signal is generatedwhen this secondary probe, labeled with dye molecules capable offluorescence resonance energy transfer, is cleaved. Cleavases havestringent requirements relative to the structure formed by theoverlapping DNA sequences or flaps and can, therefore, be used tospecifically detect single base pair mismatches immediately upstream ofthe cleavage site on the downstream DNA strand. See Ryan et al.,Molecular Diagnosis, Vol. 4, No 2, pp. 135-144 (1999); and Lyamichev etal., Nat Biotechnol., Vol. 17, pp. 292-296 (1999); see also U.S. Pat.Nos. 5,846,717 and 6,001,567 (the disclosures of which are incorporatedherein by reference in their entirety).

In some embodiments, a composition contains two or more differentlylabeled genotyping oligonucleotides for simultaneously probing theidentity of nucleotides at two or more polymorphic sites. It is alsocontemplated that primer compositions may contain two or more sets ofallele-specific primer pairs to allow simultaneous targeting andamplification of two or more regions containing a polymorphic site.

IL-1β genotyping oligonucleotides of the invention may also beimmobilized on or synthesized on a solid surface such as a microchip,bead or glass slide (see, e.g., WO 98/20020 and WO 98/20019). Suchimmobilized genotyping oligonucleotides may be used in a variety ofpolymorphism detection assays, including but not limited to probehybridization and polymerase extension assays. Immobilized IL-1βgenotyping oligonucleotides of the invention may comprise an orderedarray of oligonucleotides designed to rapidly screen a DNA sample forpolymorphisms in multiple genes at the same time.

An allele-specific oligonucleotide primer of the invention has a 3′terminal nucleotide, or preferably a 3′ penultimate nucleotide, that iscomplementary to only one nucleotide of a particular SNP, thereby actingas a primer for polymerase-mediated extension only if the allelecontaining that nucleotide is present. Allele-specific oligonucleotideprimers hybridizing to either the coding or noncoding strand arecontemplated by the invention. An ASO primer for detecting IL-1β genepolymorphisms could be developed using techniques known to those ofskill in the art.

Other genotyping oligonucleotides of the invention hybridize to a targetregion located one to several nucleotides downstream of one of the novelpolymorphic sites identified herein. Such oligonucleotides are useful inpolymerase-mediated primer extension methods for detecting one of thenovel polymorphisms described herein and therefore such genotypingoligonucleotides are referred to herein as “primer-extensionoligonucleotides”. In a preferred embodiment, the 3′-terminus of aprimer-extension oligonucleotide is a deoxynucleotide complementary tothe nucleotide located immediately adjacent to the polymorphic site.

In another embodiment, the invention provides a kit comprising at leasttwo genotyping oligonucleotides packaged in separate containers. The kitmay also contain other components, such as hybridization buffer (wherethe oligonucleotides are to be used as a probe) packaged in a separatecontainer. Alternatively, where the oligonucleotides are to be used toamplify a target region, the kit may contain, packaged in separatecontainers, a polymerase and a reaction buffer optimized for primerextension mediated by the polymerase, such as PCR. The above describedoligonucleotide compositions and kits are useful in methods forgenotyping and/or haplotyping the IL-1β gene in an individual.

As used herein, the term “haplotype with regard to the IL-1β gene” shallrefer to the haplotype consisting of the combination of thepolymorphisms at the −511 and the −31 position of the IL-1β gene andthese haplotypes shall be named in the following manner; the haplotypeshall be called “high cholesterol” if both the C to T polymorphism atthe polymorphic site −511 of the IL-1β gene (position 1423 of sequenceX04500) and the T to C polymorphism at the polymorphic site −31 of theIL-1β gene (position 1903 of sequence X04500) are present in one copy ofthe IL-1β gene. Conversely, the haplotype shall be called “lowcholesterol” if both these polymorphisms are not present in a given copyof the IL-1β gene and therefore the nucleotide at site −31 of this IL-1βgene is a T and the nucleotide at site −511 is a C in this IL-1β gene inthe chromosome referred to.

One embodiment of the genotyping method involves isolating from theindividual a nucleic acid mixture comprising the two copies of the IL-1βgene, or a fragment thereof, that are present in the individual, anddetermining the identity of the nucleotide pair at one or more of thepolymorphic sites in the two copies to assign a IL-1β genotype to theindividual. As will be readily understood by the skilled artisan, thetwo “copies” of a gene in an individual may be the same allele or may bedifferent alleles. In a particularly preferred embodiment, thegenotyping method comprises determining the identity of the nucleotidepair at each polymorphic site.

Typically, the nucleic acid mixture or protein is isolated from abiological sample taken from the individual, such as a blood sample ortissue sample. Suitable tissue samples include whole blood, semen,saliva, tears, urine, fecal material, sweat, buccal smears, skin, andbiopsies of specific organ tissues, such as muscle or nerve tissue andhair. The nucleic acid mixture may be comprised of genomic DNA,messenger polyribonucleotide (mRNA), or cDNA and, in the latter twocases, the biological sample must be obtained from an organ in which theIL-1β gene is expressed. Furthermore it will be understood by theskilled artisan that mRNA or cDNA preparations would not be used todetect polymorphisms located in introns, in 5′ and 3′ non-transcribedregions or in promoter regions. If an IL-1 gene fragment is isolated, itmust contain the polymorphic site(s) to be genotyped.

One embodiment of the haplotyping method comprises isolating from theindividual a nucleic acid molecule containing only one of the two copiesof the IL-1β gene, or a fragment thereof, that is present in theindividual and determining in that copy the identity of the nucleotideat one or more of the polymorphic sites in that copy to assign a IL-1βhaplotype to the individual. The nucleic acid may be isolated using anymethod capable of separating the two copies of the IL-1β gene orfragment, including but not limited to, one of the methods describedabove for preparing IL-1β isogenes, with targeted in vivo cloning beingthe preferred approach.

As will be readily appreciated by those skilled in the art, anyindividual clone will only provide haplotype information on one of thetwo IL-1β gene copies present in an individual. If haplotype informationis desired for the individuals other copy, additional IL-1β clones willneed to be examined. Typically, at least five clones should be examinedto have more than a 90% probability of haplotyping both copies of theIL-1β gene in an individual. In a particularly preferred embodiment, thenucleotide at each of polymorphic site is identified.

In a preferred embodiment, a IL-1β haplotype pair is determined for anindividual by identifying the phased sequence of nucleotides at one ormore of the polymorphic sites in each copy of the IL-1β gene that ispresent in the individual. In a particularly preferred embodiment, thehaplotyping method comprises identifying the phased sequence ofnucleotides at each polymorphic site in each copy of the IL-1β gene.When haplotyping both copies of the gene, the identifying step ispreferably performed with each copy of the gene being placed in separatecontainers. However, it is also envisioned that if the two copies arelabeled with different tags, or are otherwise separately distinguishableor identifiable, it could be possible in some cases to perform themethod in the same container. For example, if first and second copies ofthe gene are labeled with different first and second fluorescent dyes,respectively, and an allele-specific oligonucleotide labeled with yet athird different fluorescent dye is used to assay the polymorphicsite(s), then detecting a combination of the first and third dyes wouldidentify the polymorphism in the first gene copy while detecting acombination of the second and third dyes would identify the polymorphismin the second gene copy.

In both the genotyping and haplotyping methods, the identity of anucleotide (or nucleotide pair) at a polymorphic site(s) may bedetermined by amplifying a target region(s) containing the polymorphicsite(s) directly from one or both copies of the IL-1β gene, or fragmentthereof, and the sequence of the amplified region(s) determined byconventional methods. It will be readily appreciated by the skilledartisan that the same nucleotide will be detected twice at a polymorphicsite in individuals who are homozygous at that site, while two differentnucleotides will be detected if the individual is heterozygous for thatsite. The polymorphism may be identified directly, known aspositive-type identification, or by inference, referred to asnegative-type identification. For example, where a SNP is known to beguanine and cytosine in a reference population, a site may be positivelydetermined to be either guanine or cytosine for all individualhomozygous at that site, or both guanine and cytosine, if the individualis heterozygous at that site. Alternatively, the site may be negativelydetermined to be not guanine (and thus cytosine/cytosine) or notcytosine (and thus guanine/guanine).

In addition, the identity of the allele(s) present at any of the novelpolymorphic sites described herein may be indirectly determined bygenotyping a polymorphic site not disclosed herein that is in linkagedisequilibrium with the polymorphic site that is of interest. Two sitesare said to be in linkage disequilibrium if the presence of a particularvariant at one site enhances the predictability of another variant atthe second site. See Stevens, Mol. Diag., Vol. 4, pp. 309-317 (1999).Polymorphic sites in linkage disequilibrium with the presently disclosedpolymorphic sites may be located in regions of the gene or in othergenomic regions not examined herein. Genotyping of a polymorphic site inlinkage disequilibrium with the novel polymorphic sites described hereinmay be performed by, but is not limited to, any of the above-mentionedmethods for detecting the identity of the allele at a polymorphic site.

The target region(s) may be amplified using any oligonucleotide-directedamplification method, including but not limited to polymerase chainreaction (PCR) (U.S. Pat. No. 4,965,188), ligase chain reaction (LCR)(see Barany et al., Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 189-193(1991); and WO 90/01069), and oligonucleotide ligation assay (OLA) (seeLandegren et al., Science, Vol. 241, pp. 1077-1080 (1988)).Oligonucleotides useful as primers or probes in such methods shouldspecifically hybridize to a region of the nucleic acid that contains oris adjacent to the polymorphic site. Typically, the oligonucleotides arebetween 10 and 35 nucleotides in length and preferably, between 15 and30 nucleotides in length. Most preferably, the oligonucleotides are20-25 nucleotides long. The exact length of the oligonucleotide willdepend on many factors that are routinely considered and practiced bythe skilled artisan.

Other known nucleic acid amplification procedures may be used to amplifythe target region including transcription-based amplification systems(see U.S. Pat. Nos. 5,130,238 and 5,169,766; EP 329,822; and WO89/06700) and isothermal methods. See Walker et al., Proc. Natl. Acad.Sci. USA, Vol. 89, pp. 392-396 (1992).

A polymorphism in the target region may also be assayed before or afteramplification using one of several hybridization-based methods known inthe art. Typically, allele-specific oligonucleotides are utilized inperforming such methods. The allele-specific oligonucleotides may beused as differently labeled probe pairs, with one member of the pairshowing a perfect match to one variant of a target sequence and theother member showing a perfect match to a different variant. In someembodiments, more than one polymorphic site may be detected at onceusing a set of allele-specific oligonucleotides or oligonucleotidepairs. Preferably, the members of the set have melting temperatureswithin 5° C. and more preferably within 2° C., of each other whenhybridizing to each of the polymorphic sites being detected.

Hybridization of an allele-specific oligonucleotide to a targetpolynucleotide may be performed with both entities in solution or suchhybridization may be performed when either the oligonucleotide or thetarget polynucleotide is covalently or noncovalently affixed to a solidsupport. Attachment may be mediated, for example, by antibody-antigeninteractions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges,hydrophobic interactions, chemical linkages, UV cross-linking baking,etc. Allele-specific oligonucleotides may be synthesized directly on thesolid support or attached to the solid support subsequent to synthesis.Solid-supports suitable for use in detection methods of the inventioninclude substrates made of silicon, glass, plastic, paper and the like,which may be formed, for example, into wells (as in 96-well plates),slides, sheets, membranes, fibers, chips, dishes and beads. The solidsupport may be treated, coated or derivatized to facilitate theimmobilization of the allele-specific oligonucleotide or target nucleicacid.

The genotype or haplotype for the IL-1β gene of an individual may alsobe determined by hybridization of a nucleic sample containing one orboth copies of the gene to nucleic acid arrays and subarrays such asdescribed in WO 95/11995. The arrays would contain a battery ofallele-specific oligonucleotides representing each of the polymorphicsites to be included in the genotype or haplotype.

The identity of polymorphisms may also be determined using a mismatchdetection technique, including but not limited to the RNase protectionmethod using riboprobes (see Winter et al., Proc. Natl. Acad. Sci. USA,Vol. 82, p. 7575 (1985); and Meyers et al., Science, Vol. 230, p. 1242(1985)) and proteins which recognize nucleotide mismatches, such as theE. coli mutS protein. See Modrich, Ann. Rev. Genet, Vol. 25, pp. 229-253(1991). Alternatively, variant alleles can be identified by singlestrand conformation polymorphism (SSCP) analysis (see Orita et al.,Genomics, Vol. 5, pp. 874-879 (1989); and Humphries et al., MolecularDiagnosis of Genetic Diseases, Elles, Ed., pp. 321-340 (1996)) ordenaturing gradient gel electrophoresis (DGGE). See Wartell et at.,Nucl. Acids Res., Vol. 18, pp. 2699-2706 (1990); and Sheffield et al.,Proc. Natl. Acad. Sci. USA, Vol. 86, pp. 232-236 (1989).

A polymerase-mediated primer extension method may also be used toidentify the polymorphism(s). Several such methods have been describedin the patent and scientific literature and include the “Genetic BitAnalysis” method (“WO 92/15712) and the ligase/polymerase mediatedgenetic bit analysis (U.S. Pat. No. 5,679,524). Related methods aredisclosed in WO 91/02087, WO 90/09455, WO 95/17676, U.S. Pat. Nos.5,302,509 and 5,945,283. Extended primers containing a polymorphism maybe detected by mass spectrometry as described in U.S. Pat. No.5,605,798. Another primer extension method is allele-specific PCR. SeeRuaflo et al., Nucl. Acids Res., Vol. 17, p. 8392 (1989); Ruafio et al.,Nucl. Acids Res., Vol. 19, pp. 6877-6882 (1991); WO 93/22456; and Turkiet al., J. Clin. Invest, Vol. 95, pp. 1635-1641 (1995). In addition,multiple polymorphic sites may be investigated by simultaneouslyamplifying multiple regions of the nucleic acid using sets ofallel-specific primers as described in Wallace et al. (WO 89/10414).

In a preferred embodiment, the haplotype frequency data for eachethnogeographic group is examined to determine whether it is consistentwith Hardy-Weinberg equilibrium. Hardy-Weinberg equilibrium (see Hartlet al., Principles of Population Genomics, Sinauer Associates, 3^(rd)Edition, Sunderland, Mass. (1997), postulates that the frequency offinding the haplotype pair H₁/H₂ is equal to P_(H-W) (H₁/H₂)=2p(H₁) p(H₂) if H₁≠H₂ and P_(H-W) (H₁/H₂)=p (H₁) p (H₂) if H₁=H₂. Astatistically significant difference between the observed and expectedhaplotype frequencies could be due to one or more factors includingsignificant inbreeding in the population group, strong selectivepressure on the gene, sampling bias, and/or errors in the genotypingprocess. If large deviations from Hardy-Weinberg equilibrium areobserved in an ethnogeographic group, the number of individuals in thatgroup can be increased to see if the deviation is due to a samplingbias. If a larger sample size does not reduce the difference betweenobserved and expected haplotype pair frequencies, then one may wish toconsider haplotyping the individual using a direct haplotyping methodsuch as, for example, CLASPER System™ technology (U.S. Pat. No.5,866,404), SMD or allele-specific long-range PCR. See Michalotos-Beloinet al., Nucl. Acids Res., Vol. 24, pp. 4841-4843 (1996).

In one embodiment of this method for predicting an IL-1β haplotype pair,the assigning step involves performing the following analysis. First,each of the possible haplotype pairs is compared to the haplotype pairsin the reference population. Generally, only one of the haplotype pairsin the reference population matches a possible haplotype pair and thatpair is assigned to the individual. Occasionally, only one haplotyperepresented in the reference haplotype pairs is consistent with apossible haplotype pair for an individual, and in such cases theindividual is assigned a haplotype pair containing this known haplotypeand a new haplotype derived by subtracting the known haplotype from thepossible haplotype pair. In rare cases, either no haplotype in thereference population are consistent with the possible haplotype pairs,or alternatively, multiple reference haplotype pairs are consistent withthe possible haplotype pairs. In such cases, the individual ispreferably haplotyped using a direct molecular haplotyping method suchas, for example, CLASPER System™ technology (U.S. Pat. No. 5,866,404),SMD or allele-specific long-range PCR. See Michalotos-Beloin et al.,supra.

Glossary

-   Allele A particular form of a gene or DNA sequence at a specific    chromosomal location (locus).-   Antibodies Includes polyclonal and monoclonal antibodies, chimeric,    single-chain, and humanized antibodies, as well as Fab fragments,    including the products of an Fab or other immunoglobulin expression    library.-   Candidate gene A gene which is hypothesized to be responsible for a    disease, condition, or the response to a treatment, or to be    correlated with one of these.-   Full-genotype The unphased 5′ to 3′ sequence of nucleotide pairs    found at all known polymorphic sites in a locus on a pair of    homologous chromosomes in a single individual.-   Full-haplotype The 5′ to 3′ sequence of nucleotides found at all    known polymorphic sites in a locus on a single chromosome from a    single individual.-   Gene A segment of DNA that contains all the information for the    regulated biosynthesis of an RNA product, including promoters,    exons, introns, and other untranslated regions that control    expression.-   Genotype An unphased 5′ to 3′ sequence of nucleotide pair(s) found    at one or more polymorphic sites in a locus on a pair of homologous    chromosomes in an individual. As used herein, genotype includes a    full-genotype and/or a sub-genotype as described below.-   Genotypinq A process for determining a genotype of an individual.-   Haplotype A 5′ to 3′ sequence of nucleotides found at one or more    linked polymorphic sites in a locus on a single chromosome from a    single individual.-   Haplotype data Information concerning one or more of the following    for a specific gene: a listing of the haplotype pairs in each    individual in a population; a listing of the different haplotypes in    a population; frequency of each haplotype in that or other    populations, and any known associations between one or more    haplotypes and a trait.-   Haplotype pair Two haplotypes found for a locus in a single    individual.-   Haplotyping A process for determining one or more haplotypes in an    individual and includes use of family pedigrees, molecular    techniques and/or statistical inference.-   Homolog A generic term used in the art to indicate a polynucleotide    or polypeptide sequence possessing a high degree of sequence    relatedness to a reference sequence. Such relatedness may be    quantified by determining the degree of identity and/or similarity    between the two sequences as hereinbefore defined. Falling within    this generic term are the terms “ortholog” and “paralog”.-   Identity A relationship between two or more polypeptide sequences or    two or more polynucleotide sequences, determined by comparing the    sequences. In general, identity refers to an exact nucleotide to    nucleotide or amino acid to amino acid correspondence of the two    polynucleotide or two polypeptide sequences, respectively, over the    length of the sequences being compared.-   Isoform A particular form of a gene, mRNA, cDNA or the protein    encoded thereby, distinguished from other forms by its particular    sequence and/or structure.-   Isogene One of the isoforms of a gene found in a population. An    isogene contains all of the polymorphisms present in the particular    isoform of the gene.-   Isolated As applied to a biological molecule, such as RNA, DNA,    oligonucleotide or protein; isolated means the molecule is    substantially free of other biological molecules, such as nucleic    acids, proteins, lipids, carbohydrates, or other material, such as    cellular debris and growth media. Generally, the term “isolated” is    not intended to refer to a complete absence of such material or to    absence of water, buffers, or salts, unless they are present in    amounts that substantially interfere with the methods of the present    invention.-   Linkage Describes the tendency of genes to be inherited together as    a result of their location on the same chromosome; measured by    percent recombination between loci.-   Linkage disequilibrium Describes a situation in which some    combinations of genetic markers occur more or less frequently in the    population than would be expected from their distance apart. It    implies that a group of markers has been inherited coordinately. It    can result from reduced recombination in the region or from a    founder effect, in which there has been insufficient time to reach    equilibrium since one of the markers was introduced into the    population.-   Locus A location on a chromosome or DNA molecule corresponding to a    gene or a physical or phenotypic feature.-   Modified bases Include, e.g., tritylated bases and unusual bases,    such as inosine. A variety of modifications may be made to DNA and    RNA; thus, polynucleotide embraces chemically, enzymatically or    metabolically modified forms of polynucleotides as typically found    in nature, as well as the chemical forms of DNA and RNA    characteristic of viruses and cells. Polynucleotide also embraces    relatively short polynucleotides, often referred to as    oligonucleotides.-   Naturally-occurring A term used to designate that the object it is    applied to, e.g., naturally-occurring polynucleotide or polypeptide,    can be isolated from a source in nature and which has not been    intentionally modified by man.-   Nucleotide pair The nucleotides found at a polymorphic site on the    two copies of a chromosome from an individual.-   Ortholog A polynucleotide or polypeptide that is the functional    equivalent of the polynucleotide or polypeptide in another species.-   Paralog A polynucleotide or polypeptide that within the same species    which is functionally similar.-   Phased As applied to a sequence of nucleotide pairs for two or more    polymorphic sites in a locus, phased means the combination of    nucleotides present at those polymorphic sites on a single copy of    the locus is known.-   Polymorphic site (PS) A position within a locus at which at least    two alternative sequences are found in a population, the most    frequent of which has a frequency of no more than 99%.-   Polymorphic variant A gene, mRNA, cDNA, polypeptide or peptide whose    nucleotide or amino acid sequence varies from a reference sequence    due to the presence of a polymorphism in the gene.-   Polymorphism Any sequence variant present at a frequency of >1% in a    population. The sequence variation observed in an individual at a    polymorphic site.

Polymorphisms include nucleotide substitutions, insertions, deletionsand microsatellites and may, but need not, result in detectabledifferences in gene expression or protein function.

-   Polymorphism data Information concerning one or more of the    following for a specific gene: location of polymorphic sites;    sequence variation at those sites; frequency of polymorphisms in one    or more populations; the different genotypes and/or haplotypes    determined for the gene; frequency of one or more of these genotypes    and/or haplotypes in one or more populations; any known    association(s) between a trait and a genotype or a haplotype for the    gene.-   Polymorphism database A collection of polymorphism data arranged in    a systematic or methodical way and capable of being individually    accessed by electronic or other means.-   Polynucleotide Any RNA or DNA, which may be unmodified or modified    RNA or DNA. Polynucleotides include, without limitation, single- and    double-stranded DNA, DNA that is a mixture of single- and    double-stranded regions, single- and double-stranded RNA, and RNA    that is mixture of single- and double-stranded regions, hybrid    molecules comprising DNA and RNA that may be single-stranded or,    more typically, double-stranded or a mixture of single- and    double-stranded regions. In addition, polynucleotide refers to    triple-stranded regions comprising RNA or DNA or both RNA and DNA.    The term polynucleotide also includes DNAs or RNAs containing one or    more modified bases and DNAs or RNAs with backbones modified for    stability or for other reasons.-   Polypeptide Any polypeptide comprising two or more amino acids    joined to each other by peptide bonds or modified peptide bonds,    i.e., peptide isosteres. Polypeptide refers to both short chains,    commonly referred to as peptides, oligopeptdes or oligomers, and to    longer chains, generally referred to as proteins. Polypeptides may    contain amino acids other than the 20 gene-encoded amino acids.    Polypeptides include amino acid sequences modified either by natural    processes, such as post-translational processing, or by chemical    modification techniques that are well known in the art. Such    modifications are well described in basic texts and in more detailed    monographs, as well as in a voluminous research literature.-   Population group A group of individuals sharing a common    characteristic, such as ethnogeographic origin, medical condition,    response to treatment etc.-   Reference population A group of subjects or individuals who are    predicted to be representative of one or more characteristics of the    population group. Typically, the reference population represents the    genetic variation in the population at a certainty level of at least    85%, preferably at least 90%, more preferably at least 95% and even    more preferably at least 99%.-   Single Nucleotide Polymorphism (SNP) The occurrence of nucleotide    variability at a single nucleotide position in the genome, within a    population. An SNP may occur within a gene or within intergenic    regions of the genome. SNPs can be assayed using Allele Specific    Amplification (ASA). For the process at least 3 primers are    required. A common primer is used in reverse complement to the    polymorphism being assayed. This common primer can be between 50 and    1500 bp from the polymorphic base. The other two (or more) primers    are identical to each other except that the final 3′ base wobbles to    match one of the two (or more) alleles that make up the    polymorphism. Two (or more) PCR reactions are then conducted on    sample DNA, each using the common primer and one of the Allele    Specific Primers.-   Splice variant cDNA molecules produced from RNA molecules initially    transcribed from the same genomic DNA sequence but which have    undergone alternative RNA splicing. Alternative RNA splicing occurs    when a primary RNA transcript undergoes splicing, generally for the    removal of introns, which results in the production of more than one    mRNA molecule each of which may encode different amino acid    sequences. The term usplice variant” also refers to the proteins    encoded by the above cDNA molecules.-   Sub-genotype The unphased 5′ to 3′-sequence of nucleotides seen at a    subset of the known polymorphic sites in a locus on a pair of    homologous chromosomes in a single individual.-   Sub-haplotype The 5′ to 3′ sequence of nucleotides seen at a subset    of the known polymorphic sites in a locus on a single chromosome    from a single individual.-   Subject A human individual whose genotypes or haplotypes or response    to treatment or disease state are to be determined.-   Treatment A stimulus administered internally or externally to a    subject.-   Unphased As applied to a sequence of nucleotide pairs for two or    more polymorphic sites in a locus, unphased means the combination of    nucleotides present at those polymorphic sites on a single copy of    the locus is not known.    -   See also, Human Molecular Genetics, 2^(nd) edition. Tom Strachan        and Andrew P. Read. John Wiley and Sons, Inc. Publication, New        York,

REFERENCES CITED

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes. The discussion of references herein isintended merely to summarise the assertions made by their authors and noadmission is made that any reference constitutes prior art. Applicantsreserve the right to challenge the accuracy and pertinence of the citedreferences.

In addition, all GenBank accession numbers, Unigene Cluster numbers andprotein accession numbers cited herein are incorporated herein byreference in their entirety and for all purposes to the same extent asif each such number was specifically and individually indicated to beincorporated by reference in its entirety for all purposes.

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the invention. Many modificationsand variations of this invention can be made without departing from itsspirit and scope, as will be apparent to those skilled in the art.Functionally equivalent methods and apparatus within the scope of theinvention, in addition to those enumerated herein, will be apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications and variations are intended to fall withinthe scope of the appended claims. The present invention is to be limitedonly by the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. A method to determine the degree of serum cholesterol elevation whichwill occur in a patient during treatment with an immunosuppressantmedication comprising: a) determining for the two copies of the IL-1βgene present in the patient the identity of the nucleotide pair at thepolymorphic site −511 C→T (at position 1423 of sequence X04500) of theIL-1β gene; and b) assigning the patient to a high cholesterol elevationgroup if both pairs are AT, assigning the patient to an intermediatecholesterol elevation group if one pair is AT and one pair is GC andassigning the patient to a low cholesterol elevation group if both pairsare GC.
 2. A method to treat a patient with an immunosuppressivemedication comprising: a) determining for the two copies of the IL-1βgene present in the patient the identity of the nucleotide pair at thepolymorphic site −511 C→T (position 1423 of sequence X04500) of theIL-1β gene; and b) treating the patient with the immunosuppressionmedication if both pairs are GC and using alternative treatment if onepair is AT and one pair is GC or if both pairs are AT.
 3. The method ofclaim 2, wherein the immunosuppressive medication is selected from thelist in Table
 2. 4. The method of claim 3, wherein the immunosuppressivemedication is everolimus.
 5. The method of claim 2, wherein thealternative treatment comprises the addition of a cholesterol-loweringmedication chosen from those listed in Table
 1. 6. A method to determinethe degree of serum cholesterol elevation which will occur in a patientduring treatment with an immunosuppressant medication comprising: a)determining for the two copies of the IL-1β gene present in the patientthe identity of the nucleotide pair at the polymorphic site −31 T→C(position 1903 of sequence X04500) of the IL-1β gene; and b) assigningthe patient to a high cholesterol elevation group if both pairs are CG,assigning the patient to an intermediate cholesterol elevation group ifone pair is AT and one pair is GC and assigning the patient to a lowcholesterol elevation group if both pairs are AT.
 7. A method to treat apatient with an immunosuppressive medication comprising: a) determiningfor the two copies of the IL-1β gene present in the patient the identityof the nucleotide pair at the polymorphic site −31 T→C (position 1903 ofsequence X04500) of the IL-1β gene; and b) treating the patient with theimmunosuppression medication if both pairs are AT and using alternativetreatment if one pair is AT and one pair is GC or if both pairs are CG.8. The method of claim 7, wherein the immunosuppressive medication isselected from the list in Table
 2. 9. The method of claim 8, wherein theimmunosuppressive medication is everolimus.
 10. The method of claim 7,wherein the alternative treatment comprises the addition of acholesterol-lowering medication chosen from those listed in Table
 1. 11.A method to determine the degree of serum cholesterol elevation whichwill occur in a patient during treatment with an immunosuppressantmedication comprising: a) determining, for the two copies of thechromosome containing the IL-1β gene, present in the patient, thehaplotype with regard to the IL-1β gene and, b) assigning the patient toa high cholesterol elevation group if both said copies contain the “highcholesterol” haplotype and, c) assigning the patient to an intermediatecholesterol elevation group if one said copy contains the “highcholesterol” haplotype and one contains the “low cholesterol” haplotypeand, d) assigning the patient to a low cholesterol elevation group ifboth said copies contain the “low cholesterol” haplotype.
 12. A methodto treat a patient with an immunosuppressive medication comprising: a)determining, for the two chromosomes containing the IL-1β gene presentin the patient, the haplotype with regard to the IL-1β gene, b) treatingthe patient with the immunosuppression medication if both saidchromosomes contain the “low cholesterol” haplotype, and usingalternative treatment if one said chromosome contains the “lowcholesterol” haplotype and one contains the “high cholesterol” haplotypeor both said chromosomes contain the “high cholesterol” haplotype. 13.The method of claim 12, wherein the immunosuppressive medication isselected from the list in Table
 2. 14. The method of claim 13, whereinthe immunosuppressive medication is everolimus.
 15. The method of claim12, wherein the alternative treatment comprises the addition of acholesterol-lowering medication chosen from those listed in Table
 1. 16.The methods of claims 1 wherein the process of determining the identityof the nucleotide pair or the haplotype comprises finding SNPs anywherein the said chromosome which are in linkage disequilibrium with the −511polymorphism or the −31 polymorphism in the IL-1β gene and using therelationship of the said SNP or SNPs to determine the nature nucleotidepair or haplotype of interest.
 17. A kit for determining the nucleotidepair at the polymorphic site −511 in the IL-1β gene in a patient,comprising: a) a container containing at least one reagent specific fordetecting the nature of the nucleotide pair at the polymorphic site −511of the IL-1β gene; and b) instructions for recommended treatment optionsbased on the nature of the said nucleotide pair.
 18. A kit fordetermining the nucleotide pair at the polymorphic site −31 in the IL-1βgene in a patient, comprising: a) a container containing at least onereagent specific for detecting the nature of the nucleotide pair at thepolymorphic site −31 of the IL-1β gene; and b) instructions forrecommended treatment options based on the nature of the said nucleotidepair.
 19. A kit comprising the kits of claim 17 with instructions fordetermining the nature of the haplotype of the IL-1β gene from theresults of the above kits and instructions for recommended treatmentoptions based on the nature of the indicated haplotype.